四氢嘧啶类化合物ZL-5010抗炎免疫药理活性研究
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摘要
目的：
以丁炔二酸二乙酯、环己胺及甲醛为原料,合成四氢嘧啶类化合物ZL-5010,探讨其抗炎免疫药理活性。
方法：
以丁炔二酸二乙酯、环己胺及甲醛为原料,醋酸为催化剂,在乙醇溶剂中经氨基化作用、Mannich反应、亲核加成反应和脱氢环化反应等一步法合成多取代四氢嘧啶类化合物ZL-5010(1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯),经制备型硅胶薄层色谱分离纯化。MTT法检测正常小鼠脾细胞的代谢活力；ELISA法检测肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1 beta)的含量。采用角叉菜胶诱导的大鼠足趾肿胀模型及二甲苯诱导的小鼠耳廓肿胀模型评价化合物ZL-5010对急性炎症的抑制作用。采用醋酸诱导的小鼠扭体实验和小鼠热水浴甩尾模型观察其镇痛活性。
结果：
合成的四氢嘧啶类化合物ZL-5010为无色透明油状物质,化学式为：1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯,产率为82.7%,其结构通过'H-NMR和MS得到确证,与文献报道相符。化合物ZL-5010在20、10、5μg/ml浓度下均能抑制正常小鼠脾细胞的代谢活力,平均抑制率分别为64.5%、52.1%、18.9%,与空白对照组比较有统计学差异(P=0.000, P=0.000, P=0.036), IC50为11.95μg/ml;化合物ZL-5010 (25,12.5,6.25μg/ml)均能显著抑制正常小鼠脾细胞分泌细胞因子TNF-a,与对照组比较有统计学差异(P值均为0.000)；化合物ZL-5010在25、12.5、6.25μg/ml下均能显著抑制LPS刺激的小鼠脾细胞分泌细胞因子IL-1beta,与细菌脂多糖(LPS)组比较有统计学差异(P=0.000,P=0.000,P=0.001)；且有一定的浓度依赖趋势。化合物ZL-5010中、高剂量(100、200 mg/kg.b.w)均能明显抑制二甲苯诱导的小鼠耳廓肿胀,平均耳廓肿胀抑制率分别为42.0%、50.4%,与溶媒对照组比较有显著差异(P=0.000,P=0.000)。化合物ZL-5010高、中(200,100mg/kg)剂量组及Aspirin (200mg/kg)组大鼠平均足趾肿胀度分别为0.044ml、0.069ml、0.090ml,均低于溶媒对照组大鼠足趾肿胀度,与溶媒对照组比较有统计学意义(P=0.000,P=0.002,P=0.009)；化合物ZL-5010低剂量组大鼠平均足趾肿胀度为0.119,低于溶媒对照组,但与溶媒对照组比较无统计学差异(P=0.071)；化合物ZL-5010高剂量(200mg/kg)组在给药后3h时对大鼠足趾肿胀度的抑制率最大,最大平均抑制率为93.2%,化合物ZL-5010中剂量(100mg/kg)组在给药后3h时对大鼠足趾肿胀度抑制率最大,平均抑制率为75.0%,阿司匹林(200mg/kg)组在给药后5h时对大鼠足趾肿胀度抑制率最大,平均抑制率为64.5%。化合物ZL-5010在100、200mg/kg.b.w剂量下和阿司匹林在200 mg/kg.b.w剂量下能均可显著减少正常小鼠扭体反应次数,平均扭体反应抑制率分别为46.3%、53.5%、33.5%,与空白对照组比较有显著性差异(P=0.000,P=0.000,P=0.004)。
结论：
1.以丁炔二酸二乙酯、环己胺及甲醛为原料,经氨基化作用、Mannich反应、亲核加成反应和脱氢环化反应等多组分反应法合成了四氢嘧啶类化合物ZL-5010(1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯),方法具有操作简单,原料易得,反应条件温和,反应收率高等优点。
2.反应粗产物经制备型硅胶薄层色谱板,以正己烷、乙酸乙酯(V/V为8：1)为展开剂分离纯化,方法操作简单、易行。
3.化合物ZL-5010对正常小鼠脾细胞代谢有抑制作用,可以显著抑制正常小鼠脾细胞的代谢活力及细胞因子TNF-α的分泌,并且能够抑制LPS刺激的小鼠脾细胞分泌细胞因子IL-1beta。
4.化合物ZL-5010可以显著减少醋酸所致小鼠扭体次数,提示其具有一定的镇痛活性。
5.化合物ZL-5010可以抑制角叉菜胶诱导的大鼠足趾肿胀度及二甲苯所致的正常小鼠耳廓肿胀,提示其具有良好的抗炎作用。
6.初步证明化合物ZL-5010具有良好的抗炎镇痛活性,有待进一步开发。
Objective:
It was aimed to synthesize the tetrahydropyrimidine derivative ZL-5010 with diethyl but-2-ynedioate, cyclohexanamine and formaldehyde as starting materials and to explore anti-inflammatory and immunopharmacological activities of the compound.
Methods:
ZL-5010 (diethyl1,3- dicyclohexyl- 1,2,3,6- tetrahydropyrimidine-4,5-dicarboxylate) was synthesized from diethyl but-2-ynedioate, cyclohexanamine and formaldehyde through a domino process of one-pot multicomponent reaction, followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization. This reaction was catalyzed by acetic acid in the solvent of ethanol. ZL-5010 was separated and purified by preparative thin-layer chromatography(PTLC). The metabolic activities of normal muose splenocytes cells were determined by methylthiazolyl tetrazolium(MTT) colorimetry assay. The amounts of tumor necrosis factorα(TNF-α) and interleukin 1 (IL-1 beta) were measured with ELISA assay. Anti-inflammatory activity was estimated with two acute inflammation models, including carrageenan-induced rat-paw edema and dimethylbenzene-induced ear edema in mice. Acetic acid-induced abdominal-writhing response in mice and hot water mouse tail-flick response were used to evaluate its analgesic activity.
Results:
ZL-5010 (diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) appeared to be a colourless oil substance with a yield about 82.7%. The chemical structure of ZL-5010 was characterized by 1H-NMR and MS, which was in accord with the literature data. ZL-5010 (20,10,5μg/ml) inhibited the metabolic activity of normal mouse splenocytes, and their average inhibitive rates were 64.5%,52.1% and 18.9%, respectively, with statistically significant differences(P=0.000, P=0.000, P-0.036) as compared with control group. The half maximal inhibitory concentration (IC50) was about 11.95μg/ml. ZL-5010 (25,12.5, 6.25μg/ml) significantly decreased the production of TNF-a (P=0.000, P=0.000, P=0.000) in normal mouse splenocytes in vitro, compared with control group, and inhibited the secretion of IL-1 beta in mouse splenocytes induced by lipopolysaccharide (LPS) (P=0.000, P=0.000, P=0.001, vs LPS group). Effects of ZL-5010 on the production of IL-1 beta and TNF-a in mouse splenocytes were dose-dependent within a certain concentration range. Compared with control group, ZL-5010(100,200mg/kg.b.w)significantly reduced the dimethylbenzen-induced ear edema in normal mice(P=0.000, P=0.000), and their average inhibitive rates were 42.0% and 50.4%, respectively. ZL-5010 (100,200mg/kg) and aspirin group could significantly reduce the paw edema induced by carrageenan in rats (P=0.000, P=0.002, P=0.009 vs control group), and their average paw edema were 0.044ml、0.069ml、0.090ml, respectively. The average paw edema of ZL-5010 (50mg/kg) group was a little lower than that of control group, but there was not significance between them (P=0.071). ZL-5010 (200, 100mg/kg) reached the maximum inhibition at 3 hours after rats were administered ZL-5010, and aspirin group reached the maximum inhibition at 5 hours, their maximum inhibition rates were 75.0%,93.2% and 64.5%, respectively. ZL-5010(100,200/kg.b.w) and aspirin group significantly reduced the frequency of writhing induced by acetic acid in normal mice (P=0.000, P=0.000, P=0.001, vs control group), and their average rates of inhibition were 46.3%,53.5% and 33.5%, respectively.
Conclusions:
1.ZL-5010(diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) was successfully synthesized from diethyl but-2-ynedioate, cyclohexanamine and formaldehyde through a domino process of one-pot multicomponent reaction, followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization, which is a simple and feasible method with excellent yields.
2. ZL-5010 was purified by preparative thin-layer chromatography with N-hexane and acetoacetate (v/v=8/1) as developing solvent. The method is simple and easy to carry out.
3. ZL-5010 can significantly inhibit metabolic activity and production of TNF-a in normal mouse splenocytes, and can inhibit the secretion of IL-1 beta in mouse splenocytes induced by LPS.
4. ZL-5010 can reduce the frequency of writhing induced by acetic acid in normal mice, suggesting analgesic capacity.
5. ZL-5010 can decrease the paw edema induced by carrageenan in rats and the dimethylbenzene-niduced ear edema in normal mice, implying good anti-inflammatory activity.
6. The preliminarily experimental results demonstrate that ZL-5010 has good anti-inflammatory and analgesic activities.
以丁炔二酸二乙酯、环己胺及甲醛为原料,合成四氢嘧啶类化合物ZL-5010,探讨其抗炎免疫药理活性。
方法：
以丁炔二酸二乙酯、环己胺及甲醛为原料,醋酸为催化剂,在乙醇溶剂中经氨基化作用、Mannich反应、亲核加成反应和脱氢环化反应等一步法合成多取代四氢嘧啶类化合物ZL-5010(1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯),经制备型硅胶薄层色谱分离纯化。MTT法检测正常小鼠脾细胞的代谢活力；ELISA法检测肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1 beta)的含量。采用角叉菜胶诱导的大鼠足趾肿胀模型及二甲苯诱导的小鼠耳廓肿胀模型评价化合物ZL-5010对急性炎症的抑制作用。采用醋酸诱导的小鼠扭体实验和小鼠热水浴甩尾模型观察其镇痛活性。
结果：
合成的四氢嘧啶类化合物ZL-5010为无色透明油状物质,化学式为：1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯,产率为82.7%,其结构通过'H-NMR和MS得到确证,与文献报道相符。化合物ZL-5010在20、10、5μg/ml浓度下均能抑制正常小鼠脾细胞的代谢活力,平均抑制率分别为64.5%、52.1%、18.9%,与空白对照组比较有统计学差异(P=0.000, P=0.000, P=0.036), IC50为11.95μg/ml;化合物ZL-5010 (25,12.5,6.25μg/ml)均能显著抑制正常小鼠脾细胞分泌细胞因子TNF-a,与对照组比较有统计学差异(P值均为0.000)；化合物ZL-5010在25、12.5、6.25μg/ml下均能显著抑制LPS刺激的小鼠脾细胞分泌细胞因子IL-1beta,与细菌脂多糖(LPS)组比较有统计学差异(P=0.000,P=0.000,P=0.001)；且有一定的浓度依赖趋势。化合物ZL-5010中、高剂量(100、200 mg/kg.b.w)均能明显抑制二甲苯诱导的小鼠耳廓肿胀,平均耳廓肿胀抑制率分别为42.0%、50.4%,与溶媒对照组比较有显著差异(P=0.000,P=0.000)。化合物ZL-5010高、中(200,100mg/kg)剂量组及Aspirin (200mg/kg)组大鼠平均足趾肿胀度分别为0.044ml、0.069ml、0.090ml,均低于溶媒对照组大鼠足趾肿胀度,与溶媒对照组比较有统计学意义(P=0.000,P=0.002,P=0.009)；化合物ZL-5010低剂量组大鼠平均足趾肿胀度为0.119,低于溶媒对照组,但与溶媒对照组比较无统计学差异(P=0.071)；化合物ZL-5010高剂量(200mg/kg)组在给药后3h时对大鼠足趾肿胀度的抑制率最大,最大平均抑制率为93.2%,化合物ZL-5010中剂量(100mg/kg)组在给药后3h时对大鼠足趾肿胀度抑制率最大,平均抑制率为75.0%,阿司匹林(200mg/kg)组在给药后5h时对大鼠足趾肿胀度抑制率最大,平均抑制率为64.5%。化合物ZL-5010在100、200mg/kg.b.w剂量下和阿司匹林在200 mg/kg.b.w剂量下能均可显著减少正常小鼠扭体反应次数,平均扭体反应抑制率分别为46.3%、53.5%、33.5%,与空白对照组比较有显著性差异(P=0.000,P=0.000,P=0.004)。
结论：
1.以丁炔二酸二乙酯、环己胺及甲醛为原料,经氨基化作用、Mannich反应、亲核加成反应和脱氢环化反应等多组分反应法合成了四氢嘧啶类化合物ZL-5010(1,3-二环己基-1,2,3,6-四氢嘧啶-4,5-二甲酸二乙酯),方法具有操作简单,原料易得,反应条件温和,反应收率高等优点。
2.反应粗产物经制备型硅胶薄层色谱板,以正己烷、乙酸乙酯(V/V为8：1)为展开剂分离纯化,方法操作简单、易行。
3.化合物ZL-5010对正常小鼠脾细胞代谢有抑制作用,可以显著抑制正常小鼠脾细胞的代谢活力及细胞因子TNF-α的分泌,并且能够抑制LPS刺激的小鼠脾细胞分泌细胞因子IL-1beta。
4.化合物ZL-5010可以显著减少醋酸所致小鼠扭体次数,提示其具有一定的镇痛活性。
5.化合物ZL-5010可以抑制角叉菜胶诱导的大鼠足趾肿胀度及二甲苯所致的正常小鼠耳廓肿胀,提示其具有良好的抗炎作用。
6.初步证明化合物ZL-5010具有良好的抗炎镇痛活性,有待进一步开发。
Objective:
It was aimed to synthesize the tetrahydropyrimidine derivative ZL-5010 with diethyl but-2-ynedioate, cyclohexanamine and formaldehyde as starting materials and to explore anti-inflammatory and immunopharmacological activities of the compound.
Methods:
ZL-5010 (diethyl1,3- dicyclohexyl- 1,2,3,6- tetrahydropyrimidine-4,5-dicarboxylate) was synthesized from diethyl but-2-ynedioate, cyclohexanamine and formaldehyde through a domino process of one-pot multicomponent reaction, followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization. This reaction was catalyzed by acetic acid in the solvent of ethanol. ZL-5010 was separated and purified by preparative thin-layer chromatography(PTLC). The metabolic activities of normal muose splenocytes cells were determined by methylthiazolyl tetrazolium(MTT) colorimetry assay. The amounts of tumor necrosis factorα(TNF-α) and interleukin 1 (IL-1 beta) were measured with ELISA assay. Anti-inflammatory activity was estimated with two acute inflammation models, including carrageenan-induced rat-paw edema and dimethylbenzene-induced ear edema in mice. Acetic acid-induced abdominal-writhing response in mice and hot water mouse tail-flick response were used to evaluate its analgesic activity.
Results:
ZL-5010 (diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) appeared to be a colourless oil substance with a yield about 82.7%. The chemical structure of ZL-5010 was characterized by 1H-NMR and MS, which was in accord with the literature data. ZL-5010 (20,10,5μg/ml) inhibited the metabolic activity of normal mouse splenocytes, and their average inhibitive rates were 64.5%,52.1% and 18.9%, respectively, with statistically significant differences(P=0.000, P=0.000, P-0.036) as compared with control group. The half maximal inhibitory concentration (IC50) was about 11.95μg/ml. ZL-5010 (25,12.5, 6.25μg/ml) significantly decreased the production of TNF-a (P=0.000, P=0.000, P=0.000) in normal mouse splenocytes in vitro, compared with control group, and inhibited the secretion of IL-1 beta in mouse splenocytes induced by lipopolysaccharide (LPS) (P=0.000, P=0.000, P=0.001, vs LPS group). Effects of ZL-5010 on the production of IL-1 beta and TNF-a in mouse splenocytes were dose-dependent within a certain concentration range. Compared with control group, ZL-5010(100,200mg/kg.b.w)significantly reduced the dimethylbenzen-induced ear edema in normal mice(P=0.000, P=0.000), and their average inhibitive rates were 42.0% and 50.4%, respectively. ZL-5010 (100,200mg/kg) and aspirin group could significantly reduce the paw edema induced by carrageenan in rats (P=0.000, P=0.002, P=0.009 vs control group), and their average paw edema were 0.044ml、0.069ml、0.090ml, respectively. The average paw edema of ZL-5010 (50mg/kg) group was a little lower than that of control group, but there was not significance between them (P=0.071). ZL-5010 (200, 100mg/kg) reached the maximum inhibition at 3 hours after rats were administered ZL-5010, and aspirin group reached the maximum inhibition at 5 hours, their maximum inhibition rates were 75.0%,93.2% and 64.5%, respectively. ZL-5010(100,200/kg.b.w) and aspirin group significantly reduced the frequency of writhing induced by acetic acid in normal mice (P=0.000, P=0.000, P=0.001, vs control group), and their average rates of inhibition were 46.3%,53.5% and 33.5%, respectively.
Conclusions:
1.ZL-5010(diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) was successfully synthesized from diethyl but-2-ynedioate, cyclohexanamine and formaldehyde through a domino process of one-pot multicomponent reaction, followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization, which is a simple and feasible method with excellent yields.
2. ZL-5010 was purified by preparative thin-layer chromatography with N-hexane and acetoacetate (v/v=8/1) as developing solvent. The method is simple and easy to carry out.
3. ZL-5010 can significantly inhibit metabolic activity and production of TNF-a in normal mouse splenocytes, and can inhibit the secretion of IL-1 beta in mouse splenocytes induced by LPS.
4. ZL-5010 can reduce the frequency of writhing induced by acetic acid in normal mice, suggesting analgesic capacity.
5. ZL-5010 can decrease the paw edema induced by carrageenan in rats and the dimethylbenzene-niduced ear edema in normal mice, implying good anti-inflammatory activity.
6. The preliminarily experimental results demonstrate that ZL-5010 has good anti-inflammatory and analgesic activities.
引文
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